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Cicatrizaçao Accelerated-Mice-Skin-Acute-Wound-Healing-In-Vivo-by-Com - 2013 - Archives-of-Me

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Archives of Medical Research 44 (2013) 169e177

ORIGINAL ARTICLE
Accelerated Mice Skin Acute Wound Healing In Vivo by Combined Treatment
of Argon and Helium Plasma Needle
Elizabeth Garcıa-Alcantara,a,b Regulo L
opez-Callejas,a Pedro R. Morales-Ramırez,a
a
Rosendo Pe~na-Eguiluz, Raul Fajardo-Mu~ noz, Antonio Mercado-Cabrera,a Samuel R. Barocio,a
c

ul Valencia-Alvarado,a Benjamın G. Rodrıguez-Mendez,a Arturo E. Mu~


Ra noz-Castro,a
b a,b
Anibal de la Piedad-Beneitez, and Israel A. Rojas-Olmedo
a
Plasma Physics Laboratory and Cell Radiobiology Laboratory, Instituto Nacional de Investigaciones Nucleares, Mexico D.F., Mexico
b
Instituto Tecnologico de Toluca, Toluca, Mexico
c
Centro de Investigacion y Estudios Avanzados en Salud Animal, Facultad de Medicina Veterinaria y Zootecnia Universidad Autonoma del
Estado de Mexico, Toluca, Estado de Mexico, Mexico

Received for publication July 3, 2012; accepted January 29, 2013 (ARCMED-D-12-00341).

Background and Aims. The efficacy of a direct application of plasma needle to in vivo
wound healing was experimentally studied in mice. This kind of plasma has achieved
considerable success in blood coagulation and tissue restoration in mice. In the develop-
ment of the present study, an argon plasma needle was chosen for coagulation purposes,
whereas for healing purposes, a helium plasma needle was used.
Methods. Treatment was applied with a plasma needle produced by argon and helium to
a wound induced in laboratory mice. Tissue regeneration was carried out by three argon
plasma treatments with 0.5 SLPM flow for 1 min and three treatments of helium with
1.5 SLPM flow. Intervals between each treatment were 5 min and 60 min for argon
and helium plasmas, respectively, thus completing a total treatment time of 180 min.
Histological sections were performed to corroborate the internal bleeding and tissue
regeneration.
Results. After three treatments with argon plasma, the blood produced in the wound was
coagulated and protein material appeared. By means of treatment with helium plasma, an
approach of the wound edges was produced until the conclusion thereof. These results
were corroborated histologically.
Conclusions. This type of acceleration during the skin wound healing process can be
attributed to the formation of reactive species such as NO, which were increased in the
helium plasma needle with respect to the argon plasma needle. Ó 2013 IMSS. Published
by Elsevier Inc.
Key Words: Cold plasma, Plasma treatment, Tissue regeneration, Nitric oxide.

Introduction (1e3). Recently, the field of plasma physics has been


significantly expanding with many applications, in partic-
In biology and medicine, the term plasma refers to the
ular, plasma in medicine and as an independent medical
liquid component of the blood cells, whereas this same
field. It is emerging worldwide in a manner comparable
word in physics refers to the fourth state of matter,
to the development several years ago of laser technology
a partially ionized gas with a neutral electrical behavior
(4). Research areas such as plasma surface modification
(5), plasma bio-decontamination, and therapeutic plasma
Address reprint requests to: Regulo Lopez-Callejas, Instituto Nacional are often collectively known as plasma medicine (6). The
de Investigaciones Nucleares, Plasma Physics Laboratory, Carretera
Mexico Toluca S/N, La Marquesa, Ocoyoacac, Estado de Mexico, Mexico,
scientific basis of plasma medicine rests on a fundamental
Estado de Mexico 52750, Mexico; Phone: (þ52) (55) 5329-7200; FAX: knowledge of the plasma interaction mechanisms with
(þ52) (55) 5329-7234; E-mail: regulo.lopez@inin.gob.mx living cells and tissue (7).

0188-4409/$ - see front matter. Copyright Ó 2013 IMSS. Published by Elsevier Inc.
http://dx.doi.org/10.1016/j.arcmed.2013.02.001
170 Garcıa-Alcantara et al./ Archives of Medical Research 44 (2013) 169e177

Various biomedical applications of nonthermal atmosph- a gas nozzle and the electrode connection, producing
eric pressure plasma sources have been explored recently. an |1-mm diameter plasma spot at low power levels. The
These have been focused on the treatment of medical electrodes are fed by means of an RF voltage source at
equipment and even living tissue. A major goal of tissue 13.56 MHz as reported in Reference (23). The helium
treatment with plasmas is nondestructive surgery (8). and argon gas fluxes are controlled by means of a commer-
Provided that plasmas allow fast and efficient bacterial cial MKS mass flow controller. The power density of
inactivation, they seem suitable for the asepsis of surgical the argon and helium discharge has been measured at
tools and local tissue disinfection. However, as it is |0.2 W/cm2. Considering that the tissue is heated during
expected that a novel approach to surgery will emerge the treatment by electric power dissipation, different collat-
from plasma science, a great research effort must be made eral effects can be induced. These effects depend not only
before these techniques become common in medicine. on the reached temperature, but also on the rate and dura-
Examples of plasma applications in medicine are bacterial tion of heating. Therefore, we measured the temperature
decontamination of medical instruments (9), coating of using an infrared (IR) thermometer directly in the wound.
implants with biocompatible layers (10), surface modifica- Using this device we carried out the decontamination of
tion of substrates for cell culture (6), blood coagulation bacteria caused by S. mutans (24) and E. coli (25).
by argon plasma (6,11e15), and dermatology (7,16e19).
The most frequently used plasma devices in medical Plasma Exposure
applications are the dielectric barrier discharge (DBD)
Several series of plasma applications and different time
(8), the floating electrode dielectric barrier discharge (FE-
periods were tested at different power levels until approach-
DBD) (6,12) and the plasma needle (16). The plasma nee-
ing the optimal combination of gases (data not shown) for
dle device was originally designed for dental applications
the treatment. All treatments were carried out at room
(20). It produces a small-diameter low-power nonthermal
temperature, atmospheric pressure and according to the
atmospheric pressure glow discharge. A radiofrequency
same protocol. The experimentation was divided into two
(RF) high-voltage signal is applied to a needle-shaped
phases: the first with argon gas and the second with helium
electrode located inside a concentric gas flow nozzle with
gas. In the first case, the exposure dose was 1.2 J/cm2 and in
a diameter of a few millimeters.
the second case the exposure dose was 60 J/cm2. In the first
The plasma needle generates a multitude of reactive
phase, an argon plasma needle operating at 0.5 SLPM flow
oxygen species (ROS) together with reactive nitrogen
rate was projected onto the wound surface from a 3e5 mm
species (RNS) as well as ultraviolet (UV) radiation, and
distance and, 1 min afterward, an incisive wound was
these can reach the surface of the living tissue (6). These
inflicted. After 20 min, the process was repeated twice with
reactive species can play a key role in proliferation
the same pause time for 1 min each time. During this phase,
processes, for instance, involved in collagen production
the mouse remained slightly sedated so that a degree of
of fibroblasts and synthesis of growth factors (21,22).
coagulation, compatible with the specimen’s normal move-
The small size of the plasma needle device allows focus-
ments, was attained. From this first treatment on, it was
ing a specific treatment on spots with a few-millimeter
decided to apply a coagulation procedure. Hemorrhage
diameter without applying excessive heat to the living
must be avoided at this point (22) in order to carry out
tissue. The aim of the present paper is to report the effect
the study. Thus, in the second phase, a helium plasma
of subsequent argon and helium plasma needle treatment
needle at 1.5 SLPM was applied. After the respective
at room temperature and atmospheric pressure in regard
dose trials, a sequence of three applications, each of
to the time required for skin wound healing in mice in vivo.
5 min followed by a 60-min pause proved to be highly
successful. After each treatment the mice were released
immediately, allowing the mice normal movements and to
Experimental Set-up
lick the wound.
Plasma Source
Wound Production
A system capable of generating plasma at atmospheric
pressure by means of a torch-like device known as a plasma Balb/c (28e32 g) specimens used in the experiment were
needle (16) constituted by an assembly of elements in maintained in accordance with the Guide for the Care
a coaxial configuration has been previously developed and Use of Laboratory Animals (26). A wound (between
and reported (23). A copper filament fulfills the role of axial 6 and 10 mm long and 4- to 5-mm in depth) was inflicted
powered electrode; it is surrounded by a ceramic tube that on each rear leg of the specimen (Figure 1) with one of
provides both mechanical rigidity and electric insulation. the legs being used as a control sample. This was carried
The central electrode is visible only from the very tip of out in six mice. The wounds were exposed to the plasma
the device where the encasing tube ends. The components needle under the previously mentioned conditions and were
are assembled on a Nylamid SL frame, which includes exposed to the environment. The progress of the lesion was
Plasma Needle Application on Mice Wound 171

Figure 1. Incisions practiced on both rear legs of mice. (A color figure can be found in the online version of this article.)

followed with a microscope (50X) and photographs were Results and Discussion
taken immediately after the three argon plasma needle
Spectral Analysis
applications and after the three helium applications, with
reference to the time of the incision. Using the monochromator measurements within a 200e800
nm range, the single spectrum in Figure 2A, corresponding
Histopathological Analysis to argon flux of 0.5 SLPM, was taken. Likewise, Figure 2B
shows a typical spectrum recorded from a 1.5 SLPM of He
Tissue samples were extracted from slaughtered specimens flux, both experiments being conducted at room tempera-
in the form of slices normal to each incision trajectory, both ture. The ROS and RNS are well recognized for playing
from the treated and control mice. The slices were fixed in a dual role with both deleterious and beneficial species.
10% buffered formalin for 24 h and then processed by The respective spectra (Figure 2) shows the hydroxyl
conventional histopathological techniques. The tissue radical (OH) in both discharges, whereas nitric oxide
samples were hematoxylin-eosin stained (27) at the end, (NO) was found only in the helium discharge. NO, in
analyzed on an optical microscope and photomicrographed particular, is known to possess many important physiolog-
at 100X. In particular, for histopathological studies, six ical characteristics including, in the case of skin wounds,
mice in each test were used, i.e., six for each argon treat- that of healing by inhibiting the production of superoxide
ment time (1, 20 and 40 min) and six for each helium treat- anions (29e32).
ment time (60, 120, and 180 min). Figure 2A presents the emission bands from the argon
discharge in the wavelength range of 200e800 nm, mainly
Emission Spectroscopy of hydroxyl radical (OH) in the band of 306 nm; this is
We assembled the electromechanical spectroscopy diag-
nostic instrumentation around a controlled l-Minman mono-
chromator (Minuteman, model 305 SMP, l-Minuteman
Laboratories, Inc., Acton, MA) (28) endowed with a 0.5 m
focal length Czerny-Turner coma-corrected optical system
and interchangeable gratings. When a standard 1200
groove/mm grating is selected, the instrument exhibits
a reciprocal linear dispersion |1.67 nm/mm and a nominal
first-order half-width resolution of 0.03 nm. The grating
control consists of a DC engine-powered gearbox driving
a leadscrew, which provides 12 speeds (from 0.05e200
nm/min) and a reversible scanning mechanism. With this
grating control in operation, the monochromator range
covers from 105e1200 nm. According to the region of
energy aimed by our particular experiments (200e800
nm), an R955 photomultiplier tube (PM) with a compatible
spectral sensitivity was installed. Finally, a 300-cm long Figure 2. Emission spectra of the plasma revealing the radiation intensity
quartz optic fiber of 1-mm diameter core matched with the in the UV range from: (A) 0.5 SLPM argon flux, (B) 1.5 SLPM helium
monochromator is located in front of the plasma at |5 mm. flux. (A color figure can be found in the online version of this article.)
172 Garcıa-Alcantara et al./ Archives of Medical Research 44 (2013) 169e177

Figure 3. Coagulation process without and with argon plasma needle: (A), (B) start, (C), (E), (G) control at 1 min, 20 min and 40 min after wound, respec-
tively; (D), (F), (H) wound treated for 1 min at 1, 20 and 40 min after wound infliction, respectively, without treatment (control): (A) (C) (E) (G). With
treatment (argon): (B) (D) 0, (H). (A color figure can be found in the online version of this article.)

attributed to water vapor. Because the process is carried out 729 nm and 781 nm. These bands are in agreement with
in the atmosphere, the bands of the N2 second positive other studies (37e40). From Figures 2A and 2B, it is noted
system at 316 nm, 337 nm and 380 nm are attributed on which band NOg in helium plasmas is generated; this
to the nitrogen that is present in the discharge. The N2þ first situation can promote the wound healing process (29e41).
negative system is at 358 nm, O at 777 nm and Ar in
404 nm, 675 nm, 696 nm, 706 nm, 727 nm, 738 nm,
Temperature Monitoring
751 nm, 763 nm, 772 nm, and 794 nm; these bands
are in good agreement with previously reported data The mice wound temperature was measured with an IR
(33e36). thermometer and without treatment was close to 28 C.
As to the helium discharge (Figure 2B) in the same During the application of the plasma needle, the wound
wavelength range (200e800 nm), the emission bands are temperature increased almost instantaneously and reached
as follows: Nog third positive system at 258e259 nm, its maximum in |8e10 sec at 3e5 mm of distance between
hydroxyl radical (OH) in the band of 306 nm, the bands the plasma source and the tissue surface. In the experi-
of the N2 second positive system at 295 nm, 316 nm, ments, the plasma needle was in relative motion, and the
337 nm, 375 nm, and 380 nm, the N2þ first negative system wound temperature increased typically to 41 C and 33 C
at 358 nm, 391 nm, 427 nm, O 777 nm, as well as He at 396 for the argon and helium gases, respectively. Lower temper-
nm, 400 nm, 437 nm, 447 nm, 587 nm, 667 nm, 706 nm, atures were measured with increasing scan velocities. In
Plasma Needle Application on Mice Wound 173

all experiments, the exposure dose with argon (1.2 J/cm2) clotted blood that seals the wound (Figure 3D). Following
and helium (60 J/cm2) plasma treatments is !135 J/cm2, the second argon treatment for 1 min (Figure 3F), the dry
a dose that produces tissue damage as reported by Dobrynin crust becomes thicker and shrinks while exuding some
et al. (42). droplets of a reddish lymphatic fluid. At the third appli-
cation of 1 min, the dry crust becomes more contracted
Treatment without and with Argon Plasma Needle and covered by a number of discrete exudation points
suggesting the initiation of the tissue repairing process
We varied the experimental conditions until an optimal (Figure 3H).
condition of the wound healing was determined. Thus,
Figures 3A and 3B display incisions immediately after they
Treatment without and with Helium Plasma Needle
are carried out. The untreated control cut on the right-hand
side rear leg of each specimen exhibits profuse bleeding for Evolution of the untreated control wound is represented
|1 min after the incision (Figure 3C). At about 20 min, the in Figures 4A, 4C, 4E and 4G taken at 60, 120, 180
bleeding restricts itself to the wound borders (Figure 3E); and 240 min, respectively, after the wound infliction.
a similar condition is maintained at t 5 40 min (Figure 3G). Figure 4A reveals the presence of a thin blood clot layer
After the first 1-min treatment with argon, the blood spot covered with discrete exudation points. A more abundant
acquires a darker coloration and produces a thin dry crust of lymph secretion becomes detectable on Figure 4C at that

Figure 4. Healing asepsis process without (left-hand side) and with (right-hand side) helium plasma needle: (A), (C), (E), (G) control at 60, 120, 180 and 240
min after wound, respectively; (B), (D), (F) and (H) wound treated with helium needle plasma for 5 min at the same times after wound infliction. Without
treatment (control): (A), (C), (E), (G). With treatment (argon): (B), (D), (F), (H). (A color figure can be found in the online version of this article.)
174 Garcıa-Alcantara et al./ Archives of Medical Research 44 (2013) 169e177

time (t 5 120 min). From t 5 180 min on (Figures 4E and wound closure (29,32,42). It is possible that NO generated
4G), little significant change is apparent in the wound. Fluid by helium plasma needle has a beneficial effect on the heal-
exudates are present in all cases, although no significant ing process. Future plans are to investigate this effect.
tissue restoration becomes visible.
Histological Tests with and without Argon Plasma Needle
The helium-treated wound (Figure 4B) shows, after
60 min of its infliction, no evident evolution in tissue resto- We used six mice in each of these particular tests, either in
ration in comparison with the last argon treatment argon or helium treatments. The histopathology of the
(Figure 3H). The second helium application (Figure 4D) control wounds presented a blood clot relevant evolution
makes the wound surface ostensibly shorter and encrusted between min 1 and 40 (Figures 5A and 5C) measured with
with a thin dark blood layer. The wound width is shrunk respect to the time of the incision; 33% of the mice popu-
by healing from its borders towards the central zone. lation showed signs of blood coagulation from the first
During the third helium plasma treatment (Figure 4F), the minute on. This proportion increased to 66% at min 40,
wound borders have practically joined together with little although 100% of the cases developed hemorrhagic foci
traces of serous exudates. The tissue looks practically under the blood clots. An acute inflammatory process char-
restored, leaving only a narrow visible line even after the acterized by the presence of neutrophils took place next to
mouse leg has been flexed and extended several times. the wound borders in 86% of the population. In all cases,
From the aforementioned results, it is evident that the edges of the wounds remained constant without indica-
the helium plasma needle accelerates the wound healing tion of any small junction thereof.
process in all the treated mice. It has been shown by exper- In the case of argon plasma-treated wounds and in strict
imentation that there are effects that can modify wound heal- synchronic correspondence to the previously discussed
ing: a) ROS promotes angiogenesis that is the production of images, 100% of the treated mice showed a degree of coag-
new blood vessels or repair of existing ones (30,31,41); b) ulation (Figures 5B and 5D). The same percentage pre-
the effect of plasma screening, which promotes blood clot- sented hemorrhagic foci under the clots. A mild
ting allowing activation of platelets and formation of fibrin inflammatory reaction around the lesions occurred in 86%
filaments in blood (6,12,13). Recent research indicates that of the population. In all treated and control cases, muscle
NO promotes the acceleration of the healing process and fiber fragmentation was always present.

Figure 5. Histologic slices: (A) and (B) at 1 min, (C) and (D) at 40 min. Without treatment (control): (A) A bulging blood clot fills the cavity, separates the
edges and covers the wound (8). Under the blood clot, a small hemorrhagic point is observed (/). (C) A flat blood clot covers part of the wound surface
(8) separating the edges of it (/). With treatment (argon): (B) A concave thin blood clot covers the lesion cavity (8), which joins together the wound
borders (/). (D) A blood clot lies at the bottom of the wound (8). The wound edges are collapsed into the wound cavity coming closer across it (/).
(A color figure can be found in the online version of this article.)
Plasma Needle Application on Mice Wound 175

Figure 6. Histologic slices: (A) and (B) at 60 min, (C) and (D) at 180 min, (E) and (F) at 240 min. Without treatment (control): (A) The borders are still
separated by (/) a small blood clot that fills the wound cavity (8). (C) The wound surface is covered by abundant pale proteinaceous exudate that fills the
wound cavity and still separates the wound borders. One of them is partly collapsed toward the wound cavity (/). (E) Proteinaceous exudate without eryth-
rocytes covers the wound surface and fills the wound cavity, still separating the wound borders (/). With treatment (helium) (B) The wound borders are
partly collapsed into the wound cavity which joins them together (/). Inside the cavity, a blood clot is disintegrating (8). (D) The borders collapse
completely to the bottom of the wound cavity (/) by approximating the wound edges into the wound cavity, resulting in a macroscopic appearance of a thin
line. (F) The wound borders are completely joined, forming a flat surface and demonstrating very good tissue regeneration. (A color figure can be found in the
online version of this article.)

Histological Test without and with Helium Plasma Needle restoration (Figure 6F), which suggest that the incisions
were completely healed. Plasma needle doses of argon
The helium-treated mice exhibited, from the start, only clot
and helium have together proven to promote and/or hasten
traces in the wounds (Figures 6B, 6D, and 6F), which remain
the coagulation and healing physiological processes with
during the ensuing two applications. The occurrence of
respect to the untreated samples. Similar effects have been
hemorrhagic points decreased from 86e0%, and inflamma-
observed on FE-DBD devices (11,16,42).
tion abated along with treatment progress. A visible increase
In conclusion, we have developed a methodology for
of serous exudates lining the wounds reached 66.7% of the
in vivo coagulation (by argon plasma) and healing (by
cases 2 h after the treatment and 100% in 240 min. Only
helium plasma) of wounded mouse tissue by means of
66.7% exhibited phagocytosis of fragmentary muscle fiber.
a plasma needle, in a very short time and without any
Furthermore, the wound borders of 50% of the population
collateral damage of the tissue (Figure 4H). Wound healing
had joined together by that time while coming closer in
is a complex biological and biochemical process with
the rest of the cases. After 240 min the treated specimens
a multitude of variables involved. However, complicated
showed an almost complete lip joining, while the untreated
as the healing process may be, some facts are clear: for
ones remained without any appreciable improvement.
wound healing to occur, tissue damage during treatment
The control wounds presented (at least) one clot, one
must be minimized or eliminated while wounds must be de-
hemorrhagic point and one mild inflammatory reaction
contaminated in order to prevent any bacterial invasion.
after 240 min (Figures 6A, 6C, and 6E). There were no
Our results with sequenced argon and helium plasma nee-
cases of wound borders being reunited and 67% of the pop-
dles are consistent with previous observations using
ulation exhibited the presence of opportunistic bacterial
different plasma technologies in order to treat wounds, indi-
colonies. At the same points in time, the treated wounds
cating that non-thermal plasmas decontaminate the zone of
presented joined wound edges and normal pigmentation
176 Garcıa-Alcantara et al./ Archives of Medical Research 44 (2013) 169e177

application and allow a rapid coagulation, stimulating the 13. Fridman G, Shereshevsky A, Jost MM, et al. Floating electrode dielec-
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